Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-12-18
2002-12-03
Jones, W. Gary (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C435S091510, C435S235100, C435S375000, C536S024200, C540S465000, C540S474000
Reexamination Certificate
active
06489114
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a novel process for labeling a ribonucleic acid (RNA) with signal amplification.
BACKGROUND OF THE INVENTION
The state of the art shows that there are a large number of methods for labeling nucleotides, oligonucleotides or nucleic acids; oligonucleotides and nucleic acids will be referred to by the term polynucleotides. Polynucleotides can be labeled either during synthesis or by incorporating at least one labeled nucleotide.
A first method comprises in attaching the label to the base, whether the latter is a natural base or a modified base. A second method proposes attaching the label to the sugar, again whether the latter be a natural sugar or a modified sugar. A third method relates to attaching the label to the phosphate.
In fact, a person of skill in the art who is to label a nucleotide or a nucleotide analogue or a nucleic acid is inclined to attach the label to the base or to the sugar, which offers him more convenience and more options. This is, furthermore, what emerges from studying a large number of documents such as EP-A-0.329.198, EP-A-0.302.175, EP-A-0.097.373, EP-A-0.063.879, U.S. Pat. Nos. 5,449,767, 5,328,824, WO-A-93/16094, DE-A-3.910.151 and EP-A-0.567.841 in the case of the base or EP-A-0.286.898 in the case of the sugar. Each of these documents is hereby incorporated by reference for all purposes.
The technique of attaching the label to the phosphate is more complex especially because nucleic acids are water soluble and the reactivity of phosphate in this media is lower compared to that in organic solvents.
Even so, some documents have proposed techniques for labeling the phosphate. This applies, for example, to document EP-A-0.280.058, hereby incorporated by reference for all purposes, which describes labeling a nucleotide by attaching the label to the phosphate, with the latter being attached to the sugar in the 3′ and/or 5′ positions, when the nucleotide is a deoxyribonucleotide, and in the 2′, 3′ and/or 5′ positions when the nucleotide is a ribonucleotide. This document also describes a polynucleotide or oligonucleotide which comprises at least one labeled nucleotide as described above; this nucleotide is incorporated into the polynucleotide or oligonucleotide during synthesis.
However, the labeling strategy which is proposed by document EP-A-0.280.058 does not enable the nucleic acids to be labeled uniformly. The incorporation of the labeled nucleotides into the polynucleotides cannot be controlled; it depends entirely on the composition of synthesized polynucleotides. Thus, some polynucleotides may contain a large number of labeled nucleotides whereas others may not contain any at all. As a result, the intensity of the signal emitted by these nucleic acids will not be uniform, and therefore it will be difficult to interpret the results when detecting the nucleic acids.
In this case, the labeling is incorporated biologically without any control of the positions of the labeled nucleotides.
The document U.S. Pat. No. 5,317,098 hereby incorporated by reference for all purposes relates to nucleic acids which are labeled at their 5′ ends. This attachment uses imidazole and a linker arm. There is no associated fragmentation with the labeling. Furthermore, phosphate is added to nucleic acids and therefore kinase is used as a mean to introduce the phosphate, leading to at least one additional biological step. This document describes the labeling of a 15 mer oligonucleotide. When using large nucleic acids instead of oligonucleotide, this technique leads to the presence of a label only at the 5′ end and the specific activity of the labeled nucleic acid is low.
In addition, when the labeling is carried out on large nucleic acids without a fragmentation stage, also termed a cleavage stage, the kinetics of hybridization of these labeled nucleic acids to their complementary sequences, is slow leading to poor hybridization yield. This will therefore result in a quantitative and qualitative loss of the signal. Steric hindrance is a key factor in this reaction.
Steric hindrance may not only be the result of the length of the nucleic acid but also of the existence of secondary structures. Fragmentation helps to broke (or reduce) these structures and in this way to optimize hybridization. Steric hindrance plays a particularly important role in the case of hybridization to solid surfaces which contain a high density of capture probes, for example the DNA arrays developed by the company Affymetrix, Inc. (“Accessing Genetic Information with High-Density DNA arrays”, M. Chee et al., Science, 274, 610-614, 1996. “Light-generated oligonucleotide arrays for rapid DNA sequence analysis”, A. Caviani Pease et al., Proc. Natl. Acad. Sci. USA, 91, 5022-5026, 1994, U.S. Pat. Nos. 5,744,305, 5,445,934). Each of these references is incorporated therein by reference for all purposes. In this technology, the capture probes are generally of reduced size, being of about twenty nucleotides in length.
A large number of methods are described in the state of the art for fragmenting nucleic acids.
First, the fragmentation can be enzymatic, i.e. the nucleic acids can be fragmented by nucleases (DNases or RNases) (Methods in Enzymol., vol. 152, S. Berger and A. Kimmel, ed. Academic Press, 1987, Enzymatic techniques and Recombinant DNA Technology , <Guide to Molecular cloning >, p91-110, Molecular Cloning, a Laboratory Manual, J. Sambrook, E. F. Fritsch and T. Maniatis, Cold Spring Harbor Laboratory Press, 2
nd
Edition, p5.30-5.95, 1989). Each of these documents is hereby incorporated by reference for all purposes. Depending on the involved enzyme, this reaction generates small fragments or monomers having either a hydroxyl or a monophosphate group at their 5′-or 3′-ends.
Second, the fragmentation can be chemical. For example, in the case of DNA sequences, the depurination or depyrimidination using alkylating agents generates abasic sites which are then fragmented in the presence of a base by a mechanism termed “&bgr;-elimination” (T. Lindahl et al., Rate of Chain breakage at apurinic sites in double-stranded deoxyribonucleic acid., Biochemistry, 11, p3618-3623, 1972). The DNA's can be fragmented by oxidation, alkylation or free radical addition mechanisms, inter alia (M. Liuzzi et al., Characterization and damage in gamma-irradiated and OsO4-treated DNA using methoxyamine., Int. J. Radiat. Biol., 54, p709-722, 1988). Metal cations, which are often combined with organic molecules used as chemical catalysts, for example imidazole, are used for fragmenting RNA's. (R. Breslow and R. Xu, Recognition and catalysis in nucleic acid chemistry, Proc. Natl. Acad. Sci. USA, 90, p1201-1207, 1993. J. Hovinen et al. Imidazole Tethered oligonucleotides: Synthesis and R cleaving activity, J. Org. Chem., 60, p2205-2209, 1995). This fragmentation is preferably carried out in an alkaline medium and generates fragments having 3′-phosphate ends. Each of these documents is hereby incorporated by reference for all purposes.
However, the objective of these fragmentations is not that of facilitating or permitting labeling.
Document WO-A-88/04300 proposes a method for fragmenting and labeling RNA, using RNA molecules which possesses enzymatic properties, i.e. ribozymes. Cleavage catalysis with these ribozymes is sequence specific and the reaction yields to RNA fragments having a hydroxyl group (OH) at their 5′ end and a monophosphate at 3′ end. The labeling, which is solely radioactive labeling, is then effected by incorporating an added radioactive phosphate which is derived from a molecule of GTP. It is a phosphotransferase activity of these ribozymes category, i.e a kinase activity. The radioactive phosphate attachment is effected solely at the hydroxyl group at 5′ end and no phosphate resulting from fragmentation is used for attaching the label to RNA fragments. Furthermore, the fragmentation is only carried out by ribozymes, implying the existence of a specificity bet
Do Duc
Laayoun Ali
Miyada Charles G.
BIO Merieux
Chakrabarti Arun
Jones W. Gary
Oliff & Berridg,e PLC
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